title: Folic acid modulates eNOS activity via effects on posttranslational modifications and protein-protein interactions.
abstract: Folic acid enhances endothelial function and improves outcome in primary prevention of cardiovascular disease. The exact intracellular signalling mechanisms involved remain elusive and were therefore the subject of this study. Particular focus was placed on folic acid-induced changes in posttranslational modifications of endothelial nitric oxide synthase (eNOS). Cultured endothelial cells were exposed to folic acid in the absence or presence of phosphatidylinositol-3' kinase/Akt (PI3K/Akt) inhibitors. The phosphorylation status of eNOS was determined via western blotting. The activities of eNOS and PI3K/Akt were evaluated. The interaction of eNOS with caveolin-1, Heat-Shock Protein 90 and calmodulin was studied using co-immunoprecipitation. Intracellular localisation of eNOS was investigated using sucrose gradient centrifugation and confocal microscopy. Folic acid promoted eNOS dephosphorylation at negative regulatory sites, and increased phosphorylation at positive regulatory sites. Modulation of phosphorylation status was concomitant with increased cGMP concentrations, and PI3K/Akt activity. Inhibition of PI3K/Akt revealed specific roles for this kinase pathway in folic acid-mediated eNOS phosphorylation. Regulatory protein and eNOS protein associations were altered in favour of a positive regulatory effect in the absence of bulk changes in intracellular eNOS localisation. Folic acid-mediated eNOS activation involves the modulation of eNOS phosphorylation status at multiple residues and positive changes in important protein-protein interactions. Such intracellular mechanisms may in part explain improvements in clinical vascular outcome following folic acid treatment.
authors.affiliation: Department of Pharmacology, Therapeutics & Toxicology, Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Heath Park Campus, Cardiff CF14 4XN, UK.
authors.forename: Sarah Y
authors.initials: SY
authors.lastname: Taylor
authors.forename: Hannah M
authors.initials: HM
authors.lastname: Dixon
authors.forename: Shobana
authors.initials: S
authors.lastname: Yoganayagam
authors.forename: Natalie
authors.initials: N
authors.lastname: Price
authors.forename: Derek
authors.initials: D
authors.lastname: Lang
mesh_terms: D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology*; D005492:Folic Acid / Q000494:pharmacology*; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D013552:Swine
chemical_list: D002316:Cardiotonic Agents; D047428:Protein Kinase Inhibitors; D005492:Folic Acid; D052250:Nitric Oxide Synthase Type III; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt
keywords: Endothelium; Folic acid; Kinase; Nitric oxide synthase; Phosphorylation; Vascular biology